Lyme disease is the most common tick-borne illness in the United States and Europe. It is caused by Borrelia burgdorferi, a bacterial pathogen that is maintained in nature in a zoonotic cycle between various species of small mammals and an ixodid tick vector. A hallmark of the Lyme disease spirochete is its unusual segmented genome, which includes a large number of linear and circular plasmids. Increasing evidence indicates that plasmid-encoded functions are critical for successful adaptation to the different environments that B. burgdorferi encounters during its infectious cycle. We have developed genetic tools to investigate basic aspects of the unusual genomic organization, cellular structure and metabolism of B. burgdorferi. We have extended this investigation to an in vivo setting with an experimental system that closely mimics the natural arthropod vector/rodent host infectious cycle. Through an understanding of the basic molecular biology of the organism, we hope to gain insight into the infectious strategy utilized by this significant vector-borne pathogen and thereby facilitate efforts to prevent, diagnose and treat Lyme disease. Transmission of B. burgdorferi in nature requires ingestion of vertebrate blood by ticks, which not only acts as a means of spirochete transfer between the tick vector and vertebrate host, but also stimulates and supports spirochete replication within feeding ticks. Exposure to vertebrate blood during tick feeding also induces phenotypic changes that conditionally prime B. burgdorferi for subsequent infection of a vertebrate host. In nature, multiple strains of B. burgdorferi are stably maintained at high prevalence in both the tick vector and reservoir hosts sharing the same local geographic area. Hence during the natural enzootic cycle, infected ticks in endemic areas feed not only upon naive hosts, but also upon seropositive infected hosts. In FY2018, we considered this environmental parameter and assessed the impact of the immune status of the blood-meal host on the phenotype of the Lyme disease spirochete within the tick vector (1). The results of our study indicate that blood from an infected, immune host can have either a profoundly negative or positive impact on the virulence of the Lyme disease spirochete in feeding ticks. This dichotomous response to host blood prevents super-infection by the same B. burgdorferi strain, while promoting infection by heterologous strains, and explains how B. burgdorferi polymorphism is stably maintained within the same niche in endemic regions. Significantly, this study demonstrates for the first time that protective immunity against the Lyme disease spirochete occurs within the feeding tick vector prior to transmission, through a neutralization mechanism that does not require bacterial killing nor utilize tick midgut components. These findings are pertinent to a broad spectrum of basic and applied research endeavors in the Lyme disease field, including vaccine development, evolution and population genomics, ecology and vector biology. We previously established that a plasmid-encoded gene product, BBD18, negatively regulates RpoS, an alternative sigma factor that drives the critical global response of B. burgdorferi during host infection. However, we do not know the mechanism by which this occurs, or if BBD18 is directly or indirectly responsible for repression of RpoS. In FY2018, post-baccalaureate IRTA Britney Cheff expressed bbd18 from an inducible promoter in wild-type B. burgdorferi, demonstrated that BBD18 is required for spirochete viability, and isolated strains with compensatory mutations that permit survival without BBD18. We have conducted comparative sequence analyses of DNA and RNA from pertinent strains to gain insight into what regulates bbd18 and what else is regulated by BBD18. We have also continued a collaboration with Drs. Dulebohn and Gherardini of the Gene Regulation Section at RML, and Dr. Peter Chien at U Mass Amherst, to probe the structure of BBD18 protein and its potential interface with the protein degradation machinery of the bacterial cell. Genetic manipulation of B. burgdorferi is currently extremely inefficient, requiring microgram quantities of DNA, yet yielding only a few transformants. This severely limits the application of effective genetic screens to the Lyme disease spirochete. Endogenous plasmid-encoded restriction/modification (R/M) systems constitute part of the barrier to stable introduction of foreign DNA in B. burgdorferi. In FY2018, we extended a collaboration with Dr. Gang Fang at Mt. Sinai School of Medicine, NY, through which we have identified the DNA sequence motifs recognized by three R/M systems of the widely used B. burgdorferi type strain. Armed with this information, Dr. Jenny Wachter, a postdoctoral fellow in MGS, has designed shuttle vectors and selectable markers that lack these R/M sites. Additionally, in collaboration with Dr. Craig Martens and Stacy Ricklefs of the RTB, Jenny has completed RNA-seq analysis of strains containing or lacking R/M genes and found evidence for limited epigenetic regulation of gene expression in B. burgdorferi. Ongoing studies will assess the utility of sequence-optimized constructs for efficient transformation of B. burgdorferi and explore their potential to expand genetic studies in the Lyme disease spirochete. In FY2018 we assisted Christine Savage, a graduate student in Dr. Brian Stevenson's lab at the University of Kentucky, with an investigation of the role of a DNA- and RNA-binding protein of B. burgdorferi in the modulation of spirochete physiology (2). Our contribution to this study entailed an analysis of transcript levels in the spirochete during acquisition, persistence and transmission of B. burgdorferi by the tick vector. In March of FY2018, Dr. Rosa began a sabbatical in the laboratory of Dr. Christine Jacobs-Wagner at Yale University. During this sabbatical, Dr. Rosa is collaborating with the Jacobs-Wagner lab in an investigation of how Borrelia replicates and segregates its highly segmented genome, as required for the maintenance and survival of B. burgdorferi in nature and ultimately central to transmission of Lyme disease.